Arrowroot as a novel substrate for ethanol production by solid state simultaneous saccharification and fermentation

Ethanol production from Canna edulis Ker was successfully carried out by solid state simultaneous saccharification and fermentation. The enzymatic hydrolysis conditions of C. edulis were optimized by Plackett–Burman design. The effect of inert carrier (corncob and rice bran) on ethanol fermentation and the kinetics of solid state simultaneous saccharification and fermentation was investigated. It was found that C. edulis was an alternative substrate for ethanol production, 10.1% (v/v) of ethanol concentration can attained when 40 g corncob and 10 g rice bran per 100 g C. edulis powder were added for ethanol fermentation. No shortage of fermentable sugars was observed during solid state simultaneous saccharification and fermentation. There was no wastewater produced in the process of ethanol production from C. edulis with solid state simultaneous saccharification and fermentation and the ethanol yield of more than 0.28 tonne per one tonne feedstock was achieved. This is first report for ethanol production from C. edulis powder.     

Conversion of paper sludge to ethanol by separate hydrolysis and fermentation (SHF) using Saccharomyces cerevisiae

The conversion of ethanol from paper sludge using the separate hydrolysis and fermentation (SHF) process with cellulase and Saccharomyces cerevisiae GIM-2 were investigated in this paper. Optimization strategy based on statistical experimental designs was employed to enhance degree of saccharification by enzymatic hydrolysis of paper sludge. Based on the Plackett-Burman design, hydrolysis time, substrate concentration and cellulase dosage were selected as the most significant variable on the degree of saccharification. Subsequently, the optimum combination of the selected factors was investigated by a Box-Behnken approach. A mathematical model was developed to show the effects of each factor and their combinatorial interactions on the degree of saccharification. The optimal conditions were hydrolysis time 82.7 h, substrate concentration 40.8 g L⁻¹ and cellulase dosage 18.1 FPU g⁻¹ substrate, and a degree of saccharification of 82.1% can be achieved. When hydrolysate was further fermented with S. cerevisiae GIM-2, the conversion rate of sugar to ethanol was 34.2% and the ethanol yield was 190 g kg⁻¹ of dry paper sludge, corresponding to an overall conversion yield of 56.3% of the available carbohydrates on the initial substrate. The results derived from this study indicate that the response surface methodology is a useful tool for optimizing the hydrolysis conditions to converse paper sludge to ethanol.     

Evaluation of fuel ethanol production from aqueous ammonia-treated rice straw via simultaneous saccharification and fermentation

Rice straw (RS) has been considered a promising feedstock for ethanol production in Asia. However, the recalcitrance of biomass, particularly the presence of lignin, hinders the enzymatic saccharification of polysaccharides in RS and consequently decreases the ethanol yield. Here, we used aqueous ammonia pretreatment to remove lignin from RS (aRS). The reaction conditions were a solid:liquid ratio of 1:12, an ammonia concentration of 27% (w w⁻¹), room temperature, and a 2-week incubation. We evaluated enzymatic digestibility and the ethanol production yield. A 42% reduction in lignin content increased the glucan conversion of aRS to glucose from 20 to 71% using a combination of Cellic Ctec2 cellulases and Cellic Htec2 xylanases at enzyme loads of 15 FPU +100 XU g⁻¹ solid. Scanning electron microscopy highlighted the extensive removal of external fibres and increased porosity of aRS, which aided the accessibility of cellulose for enzymes. Using the same enzyme dosage and a solid load of 100 g L⁻¹, simultaneous saccharification and fermentation using a monoculture of Saccharomyces cerevisiae and co-culture with Candida tropicalis yielded ethanol concentrations of 22 and 25 g L⁻¹, corresponding to fermentation efficiencies of 96 and 86% fermentation, respectively. The volumetric ethanol productivities for these systems were 0.45 and 0.52 g L⁻¹ h⁻¹. However, the ethanol yield based on the theoretical glucose and xylose concentrations was lower for the co-culture (0.44 g g⁻¹) than the monoculture (0.49 g g⁻¹) due to the low xylose consumption. Further research should optimise fermentation variables or select/improve microbial strains capable of fermenting xylose to increase the overall ethanol production yield.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production

Production of reducing sugar by hydrolysis of corncob material with Streptomyces sp. cellulase and ethanol fermentation of cellulosic hydrolysate was investigated. Cultures of Streptomyces sp. T3-1 improved reducing sugar yields with the production of CMCase, Avicelase and ??-glucosidase activity of 3.8, 3.9 and 3.8 IU/ml, respectively. CMCase, Avicelase, and ??-glucosidase produced by the Streptomyces sp. T3-1 favored the conversion of cellulose to glucose. It was recognized that the synergistic interaction of endoglucanase, exoglucanase and ??-glucosidase resulted in efficient hydrolysis of cellulosic substrate. After 5 d of incubation, the overall reducing sugar yield reached 53.1 g/100 g dried substrate. Further fermentation of cellulosic hydrolysate containing 40.5 g/l glucose was performed using Saccharomyces cerevisiae BCRC 21812, 14.6 g/l biomass and 24.6 g/l ethanol was obtained within 3 d. The results have significant implications and future applications regarding to production of fuel ethanol from agricultural cellulosic waste.     

Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae

The newspaper is comprised of (w w^?1) holocellulose (70.0%) with substantial amount of lignin (16.0%). Bioconversion of the carbohydrate component of newspaper to sugars by enzymatic saccharification, and its fermentation to ethanol was investigated. Of various enzymatic treatments using cellulase, xylanase and laccase, cellulase enzyme system was found to deink the newspaper most efficiently. The saccharification of deinked paper pulp using enzyme cocktail containing exoglucanase (20 U g^?1), β-glucosidase (60 U g^?1) and xylanase (80 U g^?1) resulted in 59.8% saccharification. Among additives, 1% (v v^?1) Tween 80 and 10 mol m^?3 CoCl₂ improved the enzymatic hydrolysis of newspaper maximally, releasing 14.64 g L^?1 sugars. The fed batch enzymatic saccharification of the newspaper increased the sugar concentration in hydrolysate from 14.64 g L^?1 to 38.21 g L^?1. Moreover, the batch and fed batch enzymatic hydrolysates when fermented with Saccharomyces cerevisiae produced 5.64 g L^?1 and 14.77 g L^?1 ethanol, respectively.     

Optimization of concomitant production of cellulase and xylanase from Rhizopus oryzae SN5 through EVOP-factorial design technique and application in Sorghum Stover based bioethanol production

Current study deals with the production of cellulases and xylanases from the Rhizopus oryzae SN5 isolated from composed soil of Himalayan pine forest, in order to meet the challenges of lignocellulosic biomass based biorefineries. Culture parameters for concomitant production of cellulase and xylanase were optimized through EVOP-factorial design technique under solid state fermentation. And maximum yield of cellulase and xylanase were obtained 437.54 U/gds and 273.83 U/gds, respectively at 30 °C and pH 6.0 after 5 days of incubation. On applying these enzymes for the saccharification of the dilute acid pretreated Sorghum Stover (SS), 0.407 g/g sugar was yielded. This hydrolysate on fermentation, yielded 0.411 g/g ehanol with Saccharomyces cerevisiae (NCIM 3288), which could be considered a good conversion. Therefore, Rhizopus oryzae SN5 was found as potent strain for the production of the cocktail of lignocellulosic biomasss hydrolytic enzymes and would be promising tool in the area of lignocellulose based bio-refineries.     

Biochemical conversion of sugarcane bagasse into bioproducts

The ground sugarcane bagasse conversions were examined through chemical treatment methods employing soaking in aqueous ammonia (SAA), and ethyl-hydro-oxides (EHOs). To characterize a chemical treatment method, both generated solvent based extract and pulp were examined. The generated pulps were evaluated through chemical composition and enzymatic saccharification. The enzyme mixtures were investigated including Trichoderma reesei Rut C-30 originated cellulase, T. reesei Rut C-30 originated cellulase with external added β-glucosidase, Accellerase^® 1500, and Cellic^® CTec2. The physiochemical effects of chemical treatments on the structural-chemical properties of treated-bagasse were also analyzed at high substrate enzymatic saccharification. The substrate loadings (using both SAA-treated and EHOs-treated bagasse) of 125, 150, 175, 200, and 225 g L⁻¹ were examined during enzymatic saccharification process. The generated phenolic compounds were characterized based on density, antioxidant activity, and anticancer activity. All findings are discussed in relation to developing a self-sustainable integrated biorefinery.     

Enhanced saccharification of SO2 catalyzed steam-exploded corn stover by polyethylene glycol addition

Corn stover is a renewable, low cost and abundant feedstock in China. Its effective utilization is crucial for providing bioenergy, releasing environmental pollution and increasing farmers’ income. This aim of this study was to obtain the efficient saccharification of SO₂ catalyzed steam-exploded corn stover (SSECS) by polyethylene glycol (PEG) addition. According to the results, adding PEG6000 could lower the enzyme loading by 33.3%. With 20% solid loading, the highest glucose concentration of 102 g L⁻¹ and 91.3% saccharification yield were obtained using 30 CBU (g glucan)⁻¹ ??-glucosidase and 10 FPU (g glucan)⁻¹ cellulase in presence of PEG6000. In addition, protein and enzyme activities assays in the supernatants revealed that PEG could facilitate the desorption of enzyme protein from lignocellulose. These indicated that PEG addition not only can enhance enzymatic saccharification at high substrate concentration, but also can improve enzyme recycling by reducing the enzyme activity loss caused by adsorption during the hydrolysis.     

Dilute oxalic acid pretreatment for biorefining giant reed (Arundo donax L.)

Biomass pretreatment is essential to overcome recalcitrance of lignocellulose for ethanol production. In the present study we pretreated giant reed (Arundo donax L.), a perennial, rhizomatous lignocellulosic grass with dilute oxalic acid. The effects of temperature (170-190 °C), acid loading (2-10% w/w) and reaction time (15-40 min) were handled as a single parameter, combined severity. We explored the change in hemicellulose, cellulose and lignin composition following pretreatment and glucan conversion after enzymatic hydrolysis of the solid residue. Two different yeast strains, Scheffersomyces (Pichia) stipitis CBS 6054, which is a native xylose and cellobiose fermenter, and Saccharomyces carlsbergensis FPL-450, which does not ferment xylose or cellobiose, were used along with commercial cellulolytic enzymes in simultaneous saccharification and fermentation (SSF). S. carlsbergensis attained a maximum ethanol concentration of 15.9 g/l after 48 h at pH 5.0, while S. stipitis, at the same condition, took 96 h to reach a similar ethanol value; increasing the pH to 6.0 reduced the S. stipitis lag phase and attained 18.0 g/l of ethanol within 72 h.     

Feasibility of reed for biobutanol production hydrolyzed by crude cellulase

The aim of this study was to efficiently utilize reed for both cellulase and biobutanol production. The unprocessed cellulase blend produced under solid-state fermentation using reed as the substrate showed a similar reducing sugar yield using Whatman filter paper to the commercial enzyme blend (38.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (29.3%–14.6%) and lignin (17.2%–14.1%) content and increase cellulose content (42.5%–62.3%) from reed. Enzymatic hydrolysis of organosolv-pretreated reed using the crude cellulase with enzyme loading of 25 FPU/g reed, 20% solid content at 50 °C and pH 5.5 resulted in a reed hydrolysate containing 40.01 g/L glucose and 3.55 g/L xylose after 72 h. Fermentation of the hydrolysate medium by Clostridium acetobutylicum produced 9.07 and 14.24 g/L of biobutanol and ABE with yield of 0.21 g/g and 0.33 g/g, respectively. This study proved that crude cellulase complex produced under solid state fermentation and organsolv pretreatment can efficiently provide reed hydrolysate that can be converted to biobutanol without any commercial cellulase usage.     

Effect of nutrient supplementation on ethanol production in different strategies of saccharification and fermentation from acid pretreated rice straw

The effect of nutrient supplementation on ethanol production by recently selected thermotolerant yeast (Kluyveromyces marxianus NRRL Y-6860) was investigated in different strategies of saccharification and fermentation employing rice straw pretreated by dilute acid. Among the evaluated strategies, similar ethanol yields (Y_P/S ∼ 0.23 g g⁻¹) were obtained with or without nutrient addition. However, considering the whole process time, the strategy based on simultaneous saccharification and fermentation (SSF), without pre-hydrolysis, was assigned as the most suitable configuration due to the highest ethanol volumetric productivity (1.4 g L⁻¹ h⁻¹), about 2-fold higher in relation to the others. The impact of enzymatic preparation employed in this study was also evaluated on glucose fermentation in semi-synthetic medium. The enzymatic preparation affected both glucose consumption and ethanol production by K. marxianus NRRL Y-6860, but just in the absence of nutrients. Therefore, the enzyme type and loading should be carefully defined, not only by the capital costs involved, but also by the possibility of increasing the fermentation inhibitors.     

Aerobic and anaerobic sequential culture fermentation (AASF) to produce bio-hydrogen from steam-exploded cornstalk

A novel aerobic and anaerobic sequential culture fermentation (AASF) method was designed to improve the conversion efficiency of steam-exploded cornstalk during bio-hydrogen production. The enzyme activities of cellulase and β-glucosidase produced by Trichoderma viride ACCC 30169 were 76.79 FPU g⁻¹ dry weight and 45.23 IU g⁻¹ dry weight after 6-days steam-exploded cornstalk fermentation, respectively. The aerobic fermentation residue was used as the substrate for bio-hydrogen production by Clostridium butyricum AS1.209 anaerobic fermentation. The optimum solid-to-liquid ratio of the anaerobic fermentation substrate was 1:5. The maximum bio-hydrogen yield was attained on the medium with addition of 0.1 g g⁻¹ substrate urea after 2 days of aerobic fermentation. Compared with simultaneous saccharification and fermentation (SSF), AASF for bio-hydrogen production could shorten the fermentation period by at least 66% and the hydrogen yield reached 83% of the total volume after 24 h of anaerobic fermentation. AASF from steam-exploded cornstalk was an effective way for bio-hydrogen production without additional commercial cellulase.     

Sweet sorghum as a whole-crop feedstock for ethanol production

The potential of sweet sorghum as an alternative crop for ethanol production was investigated in this study. Initially, the enzymatic hydrolysis of sorghum grains was optimized, and the hydrolysate produced under optimal conditions was used for ethanol production with an industrial strain of Saccharomyces cerevisiae, resulting in an ethanol concentration of 87 g L⁻¹. From the sugary fraction (sweet sorghum juice), 72 g L⁻¹ ethanol was produced. The sweet sorghum bagasse was submitted to acid pretreatment for hemicellulose removal and hydrolysis, and a flocculant strain of Scheffersomyces stipitis was used to evaluate the fermentability of the hemicellulosic hydrolysate. This process yielded an ethanol concentration of 30 g L⁻¹ at 23 h of fermentation. After acid pretreatment, the remaining solid underwent an alkaline extraction for lignin removal. This partially delignified material, known as partially delignified lignin (PDC), was enriched with nutrients in a solid/liquid ratio of 1 g/3.33 mL and subjected to simultaneous saccharification and fermentation (SSF) process, resulting in an ethanol concentration of 85 g L⁻¹ at 21 h of fermentation. Thus, from the conversion of starchy, sugary and lignocellulosic fractions approximately 160 L ethanol.ton⁻¹ sweet sorghum was obtained. This amount corresponds to 13,600 L ethanol.ha⁻¹.     

Ethanol production from agroindustrial biomass using a crude enzyme complex produced by Aspergillus niger

This study investigates ethanol production from simultaneous fermentation and saccharification (SFS) and separated hydrolysis and fermentation (SHS) using enzyme complexes produced by Aspergillus niger strains (ATCC 16404, ATCC 1057, ATCC 9029). The enzyme complexes were produced by solid-state fermentation (SSF) on inexpensive and readily available agroindustrial products: rice byproduct (composed of AFEX-treated rice rust and rice bran), whey and sugarcane bagasse. The ethanol was produced by Saccharomyces cerevisiae Y904 using whey and rice byproduct as the substrate and the enzyme complex produced by A. niger. The best result for solid-state fermentation (40 U/g of dry substrate, A. niger ATCC 16404) was obtained in a 0.5 L rotating drum bioreactor at 40 °C filled half filled with solid biomass composed of rice byproduct (86% wt/wt), whey (12% wt/wt) and CaCl₂ (2.0% wt/wt). The best result for ethanol fermentation (11.7 g/L of ethanol) was obtained after 12 h of SFS at pH 4.5 and 35 °C. A comparative study of ethanol production by Trichoderma reesei CCT 2768 and A. niger ATCC 16404 complexes under the same optimised SFS and SSF conditions was also performed, revealing that ethanol production by the A. niger enzyme complex was 2.25 times higher than that by T. reesei. These findings suggest that the ethanol production using crude enzymatic complexes produced by A. niger and agroindustrial biomass described in this paper is very promising in terms of disposal of the whey produced by cheese-making and other dairy food processing.     

Comparative study on ethanol production from pretreated sugarcane bagasse using immobilized Saccharomyces cerevisiae on various matrices

Microwave alkali pretreated sugarcane bagasse was used as a substrate for production of cellulolytic enzymes, needed for biomass hydrolysis. The pretreated sugarcane bagasse was enzymatic hydrolyzed by crude unprocessed enzymes cellulase (Filter paper activity 9.4 FPU/g), endoglucanase (carboxymethylcellulase, 148 IU/g), β-glucosidase (116 IU/g) and xylanase (201 IU/g) produced by Aspergillus flavus using pretreated sugarcane bagasse as substrate under solid state fermentation. Concentrated enzymatic hydrolyzate was used for ethanol production using Saccharomyces cerevisiae immobilized on various matrices. The yield of ethanol was 0.44 g_p/g_s in case of yeast immobilized sugarcane bagasse, 0.38 g_p/g_s using Ca-alginate and 0.33 g_p/g_s using agar-agar as immobilization matrices. The immobilized yeast studied up to 10 cycles in case of immobilized sugarcane bagasse and up to 4 cycles in case of agar-agar and calcium alginate for ethanol production under repeated batch fermentation study.     

Enzymatic hydrolysis and fermentation of crushed whole sugar beets

Pectinase and cellulase enzymes were used for hydrolysis of whole sugar beets and the hydrolyzates were fermented with Escherichia coli KO11 and Saccharomyces cerevisiae via simultaneous saccharification and fermentation (SSF). Ethanol production rate was significantly higher for S. cerevisiae than for E. coli KO11. The combined effect of pectinase and cellulase loadings on ethanol production as well as residual galacturonic acid and arabinose concentrations were modeled for fermentations with S. cerevisiae. Ethanol yields of more than 92% were reached with moderate to high cellulase and pectinase loadings at 0.51 FPU g⁻¹ and 51 U g⁻¹ of dry biomass, respectively. Ethanol yields of 85% were achieved without any enzyme addition. However, addition of cellulase and pectinase enzymes increased effluent arabinose and galacturonic acid concentrations and reduced total suspended solids. This study demonstrated the yield potential of fermentation of crushed, whole sugar beets with or without the addition of cellulase and pectinase enzymes.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Optimization of two-stage fractionation process for lignocellulosic biomass using response surface methodology (RSM)

A two-stage process using aqueous ammonia and hot-water has been investigated to fractionate corn stover. To maximize hemicelluloses recovery and purity in the liquid hydrolyzate by optimizing the fractionation process, the experiments were carried out employing response surface methodology (RSM). A central composite design (CCD) was used to evaluate and confirm the effectiveness and interactions of factors. The optimal fractionation conditions were determined to be as follow: (1) First-stage reactor operated in batch mode using a 15% NH₄OH solution (w_NH3 = 15%) at 1:10 solid:liquid ratio, 60 °C, and 24 h; (2) second stage percolation reactor operated using hot-water at 20 cm³ min⁻¹, 200 °C, and 10 min.The model predicted 51.5% xylan recovery yield and 82.4% xylan purity under these conditions. Experiments confirmed the maximum xylan recovery yield and purity were 54.7% and 83.9% respectively under the optimal reaction conditions.With the solids resulting from the two-stage treatment, 87%-98% glucan digestibilities were obtained with 15 FPU of GC 220 per g-glucan and 30 CBU of Novo 188 per g-glucan enzyme loadings. Xylan digestibility of xylooligomer hydrolysates reached 76% with 8000 GXU per g-xylan of Multifect-Xylanase loading. In the simultaneous saccharification and fermentation (SSF) test using treated solids and Saccharomyces cerevisiae (D₅A), 86 % to 98% of ethanol yield was obtained on the basis of the glucan content in the treated solids.     

Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production

A newly isolated soil fungus, Penicillium sp. TG2, had cellulase activities that were comparable to those of Trichoderma reesei RUT-C30, a common commercial strain used for cellulase production. The maximal and specific activities were 1.27 U/mL and 2.28 U/mg for endoglucanase, 0.31 U/mL and 0.56 U/mg for exoglucanase, 0.54 U/mL and 1.03 U/mg for β-glucosidase, and 0.45 U/mL and 0.81 U/mg for filter paper cellulase (FPase), respectively. Optimal FPase activity was at pH 5.0 and 50 °C. We used a simultaneous saccharification and fermentation (SSF) process, which employed the yeast Kluyveromyces marxianus and Penicillium sp. TG2 cellulolytic enzymes, to produce ethanol from empty palm fruit bunches (EFBs), a waste product from the palm oil industry. The present findings indicate that Penicillium sp. TG2 has great potential as an alternative source of enzymes for saccharification of lignocellulosic biomass.